Azithromycin and derivatives as antiprotozoal agents

ABSTRACT

A method of use of azithromycin or derivatives of azithromycin in the treatment of infection caused by Toxoplasma gondii in mammals is disclosed.

BACKGROUND OF THE INVENTION

The present invention is directed to the use of compounds of the formula(I) as defined below, viz., azithromycin, its 4"-epimer, andcorresponding 4"-deoxy-4"-amino analogs in the treatment of systemicprotozoal infections in mammals, particularly in the treatment oftoxoplasmosis, a protozoal infection due to strains of Toxoplasmagondii, particularly troublesome in pregnant women and among those suchas AIDS patients, who are immune deficient.

Azithromycin is the U.S.A.N. (generic name) for9a-aza-9a-methyl-9-deoxo-9a-homoerythromycin A, a broad spectrumantibacterial compound derived from erythromycin A. Azithromycin wasindependently discovered by Bright, U.S. Pat. No. 4,474,768 and Kobrehelet al., U.S. Pat. No. 4,517,359. The name"N-methyl-11-aza-10-deoxo-10-dihydroerythromycin A" was employed inthese patents. The present more systematic name is based upon the ringexpansion and replacement nomenclature of the "IUPAC Nomenclature ofOrganic Chemistry, 1979 Edition," Pergamon Press, 1979, pp. 68-70, 459,500-503. 4"-Epi-azithromycin(4"-epi-9a-aza-9a-methyl-9-deoxo-9a-homoerythromycin A),4"-amino-4"-deoxy-azithromycin(4"-amino-9a-aza-9a-methyl-9-deoxo-4"-deoxy-9a-homoerythromycin A), and4"epi-4"-amino-4"-deoxyazithromycin A(4"-epi-4"-amino-9a-aza-9a-methyl-9-deoxo-4"-deoxy-9a-homoerythromycinA), also broad spectrum antibacterials derived from erythromycin A, arethe subjects of Bright, U.S. Pat. No. 4,526,889, Hauske and Nagel, U.S.Pat. No. 4,512,982, and Hauske and Nagel, loc. cit., respectively.

There is a continuing need for drugs which are effective againstprotozoal infections in mammals, in particular against toxoplasmosis inman. Transmission of the disease may occur transplacentally, byingestion of raw or undercooked meat containing tissue cysts, or byexposure to oocysts in cat feces. Neonatal congenital toxoplasmosis,which is acquired transplacentally, the mother having acquired a primaryinfection during or prior to pregnancy, can lead to spontaneousabortion, miscarriage or still-birth, birth defects, or the birth of achild with the clinical disease. The disease can cause brain damage andeven death in those having weakened immune systems, particularly amongthose suffering from AIDS (acquired immune deficiency syndrome) wheretoxoplasma encephalitis is a commonly found, life threatening infection.Heretofore, there has been no alternative to the present regimen ofpyrimethamine plus a sulfonamide--a relatively toxic regimen withnumerous side effects among the latter patient population. Approximately20% of AIDS patients are seropositive for Toxoplasma antibodies andapproximately 30% of these seropositive individuals will suffertoxoplasmic encephalitis, reflecting the critical problem in thispatient population. In one recent series, approximately 50% of thepatients died, median time to death being 4 months. Furthermore, sincethe incidence of relapse is also prohibitively high, new drugs areneeded which can be given both for initial treatment and as suppressivetherapy for the life of the patient.

It has recently been reported that the macrolide antibiotic,roxithromycin (the 9-[O-(2-methoxyethoxymethyl)]oxime of erythromycin A)possesses activity against toxoplasmosis in mice (see Hofflin andRemington, Antimicrobial Agents and Chemotherapy, vol. 31, pp. 346-348(1987); and leading references there cited). ##STR1## (Ia) R¹ =OH, R² =Hazithromycin (Ib) R¹ =H, R² =OH 4"-epi-azithromycin

(Ic) R¹ =NH₂, R² =H 4"-amino-4"-deoxy-azithromycin

(Id) R¹ =H, R² =NH₂ =4"-epi-4"-amino-4"-deoxy-azithromycin

SUMMARY OF THE INVENTION

We have now found that the compounds of the formula (I), wherein one ofR¹ and R² is hydrogen and the other is hydroxy or amino (convenientlynamed herein as azithromycin derivatives, vide supra) possess remarkablypotent activity against Protozoa, particularly Toxoplasma species, andso are valuable in pharmaceutical compositions for a method of treatingor preventing protozoal infections in mammals, including man. Thesecompounds are especially valuable in the treatment of toxoplasmosis, aninfection due to a strain of Toxoplasma gondii, which, as noted above,is a particular problem in pregnant women and in immune compromisedpatients.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is readily carried out. The compounds of theformula (I) are prepared according to the methods of U.S. Pat. Nos.4,474,768, 4,512,982 and 4,526,889, cited above, which are herebyincluded by reference. A particularly valuable form of azithromycin (Ia)for this purpose is azithromycin dihydrate prepared according to methodsdisclosed in Examples below.

The utility of the compounds of the formula (I) in the treatment orprevention of protozoal infections in mammals is demonstrated by theirremarkable activity in model Toxoplasma gondii infections in mice. Forexample, we have found azithromycin (Ia) to have potent in vivo activityagainst murine toxoplasmosis. Mice infected intraperitoneally with 10²tachyzoites of the virulent RH strain of T. gondii and treated 24 hourslater with 200 mg azithromycin kg/day orally by gavage (solubilized inpolyethylene glycol 200) for 10 days all survived. Concentrations of 100or 50 mg/kg resulted in 80 and 20% survival, respectively. Furtherexperiments revealed that one daily dose of 200 mg/kg for each of 3 daysafter infection resulted in 100% survival of mice infected with 10³ RHtachyzoites. Moreover, this concentration of the drug protected 100% ofinfected mice when administered as late as 72 hours after infection with10² RH tachyzoites. Additional experiments revealed that 70% of miceinfected intracerebrally with 10⁴ tachyzoites of the C56 strain of T.gondii and treated with 200 mg/kg/day for 10 days survived, but only 10%of untreated controls survived. These results indicate that azithromycinis highly effective against infection with T. gondii. (See Hofflin etal., cited above, and references there cited, for more detaileddescriptions of these murine toxoplasmosis models).

In the treatment or prevention of systemic protozoal infections inmammals, particularly toxoplasmosis in man due to strains of Toxoplasmagondii, the compounds of the formula (I), including the pharmaceuticallyacceptable salts thereof, are dosed orally or parenterally. Oral dosagewill generally be preferred, particularly in cases where the drug isdosed chronically as a preventive measure. However, particularly inacute administration for severe cases of toxoplasmosis, parenteraladministration may be preferred, a matter to be determined at thediscretion of the attending physician. The preferred dosage range isabout 5-100 mg per kg of body weight per day, in single or divided dailydoses, regardless of the route of administration. In special situations,particularly in life-threatening cases of infection, higher doses may beprescribed at the discretion of the attending physician.

When used to treat or prevent a systemic protozoal infection in amammal, particularly toxoplasmosis in man, the compounds of the formula(I), including the pharmaceutically acceptable salts thereof, can bedosed alone, but are preferably dosed in the form of pharmaceuticalcompositions comprising the active compound and apharmaceutically-acceptable carrier or diluent. Such pharmaceuticalcompositions, whether for oral or parenteral administration, areprepared according to conventional methods of pharmacy, for example, asdisclosed in U.S. Pat. Nos. 4,474,768, 4,512,982 and 4,526,889, citedabove, and included by reference.

The present invention is illustrated by the following example, but isnot limited to the details thereof.

EXAMPLE 1 Non-Hygroscopic Azithromycin Dihydrate Method A

The hygroscopic monohydrate of Preparation 1 (100 g;water-content:3.1%), tetrahydrofuran (220 ml) and diatomaceous earth (5g) were combined in a 500 ml Erlenmyer flask, stirred for 30 minutes andfiltered with 20 ml of tetrahydrofuran wash. The combined filtrate andwash was transferred to a 3 liter round bottom flask. The solution wasstirred vigorously and H₂ O (2.0 ml) was added. After 5 minutes, hexane(1800 ml) was added over 5 minutes, with continued vigorous stirring.Following an 18 hour granulation period, title product was recovered byfiltration with 1×10 ml hexane wash, and dried in vacuo to 4.6±0.2% H₂ Oby Karl Fischer, 89.5 g.

Method B

The hygroscopic monohydrate of Preparation 1 (197.6 g) andtetrahydrofuran (430 ml) were charged to a reactor and the mixturestirred to achieve a milky white solution. Activated carbon (10 g) anddiatomaceous earth (10 g) were added and the mixture stirred for 15minutes, then diluted with 800 ml of hexane and filtered with suctionover a pad of diatomaceous earth with 250 ml of hexane for wash. Thecombined filtrate and wash was diluted to 2500 ml with hexane and warmedto 34° C. With stirring, 24.7 ml of H₂ O was added. The mixture wasallowed to cool to room temperature, granulated for five hours and titleproduct recovered and dried as in Method A, 177.8 g.

The dihydrate melts sharply at 126° C. (hot stage, 10°/minute);differential scanning calorimetry (heating rate, 20° C./minute) shows anendotherm at 127° C.; thermal gravimetric analysis (heating rate 30°C./minute) shows a 1.8% weight loss at 100° C. and a 4.3% weight loss at150° C.; ir (KBr) 3953, 3553, 3488, 2968, 2930, 2888, 2872, 2827, 2780,2089, 1722, 1664, 1468, 1426, 1380, 1359, 1344, 1326, 1318, 1282, 1270,1252, 1187, 1167, 1157, 1123, 1107, 1082, 1050, 1004, 993, 977, 955,930, 902, 986, 879, 864, 833, 803, 794, 775, 756, 729, 694, 671, 661,637, 598, 571, 526, 495, 459, 399, 374, 321 and 207 cm⁻¹ ; [alpha]_(D)²⁶ =-41.4° (c=1, CHCl₃)

Anal Calcd. for C₃₈ H₇₂ N₂ O₁₂.2H₂ O:

C, 58.14; H, 9.77; N, 3.57; OCH₃, 3.95; H₂ O, 4.59.

Found:

C, 58.62; H, 9.66; N, 3.56; OCH₃, 4.11; H₂ O, 4.49.

Neutralization Equivalent (0.5N HCl in 1:1 CH₃ CN:H₂ O):

Calcd.: 374.5. Found: 393.4.

Samples of a dihydrate, slightly over dried to contain 4.1% water (lessthan theoretical) rapidly picked-up water at 33%, 75% or 100% relativehumidities to achieve the theoretical water content (4.6%) for thedihydrate. At 33% and 75% relative humidities, water content remainedessentially constant for at least 4 days. At 100% relative humidity, thewater content further rose to about 5.2, where it remained essentiallyconstant of the next three days.

A sample of the same dihyrate, maintained at 18% relative humiditygradually lost water. At four days, the water content was 2.5% and at 12days, 1.1%.

EXAMPLE 2 Azithromycin Powder for Oral Suspension

The following powdered ingredients were thoroughly blended:

    ______________________________________                                        Azithromycin Dihydrate 1268.5 g                                               (1200 g on anhydrous basis)                                                   Sucrose                 23000 g                                               Sodium phosphate         250 g                                                tribasic dodecahydrate                                                        Sodium benzoate           90 g                                                Hydroxypropylcellulose    40 g                                                Xanthan gum               40 g                                                Certified food coloring                                                                                 3 g or as                                           agent(s) in solid form required to                                                                   achieve the                                                                   desired color                                          Fruit and/or vanilla     440 g or as                                          flavoring agents in    required to                                            solid form             achieve the                                                                   desired taste                                          ______________________________________                                    

The resulting blend contains 47.75 mg of azithromycin activity per gram.Amber screw cap bottles (60 ml) are filled with 10.47 g of the blend.Prior to oral administration as a suspension, distilled water is added(25 ml) and the mixture shaken. One teaspoon (5 cc) of this mixtureprovides a 100 mg dose of azithromycin. Higher or lower doses areachieved by appropriate modification of the dosage volume.

EXAMPLE 3 Azithromycin Capsules (250 mg) for Oral Administration

The following ingredients were accurately weighed, combined, and blendedin a suitable blender for 15 minutes.

    ______________________________________                                        Hydrated azithromycin                                                                            3360.9 g*                                                  Anhydrous lactose  2015.9 g                                                   Corn starch        611.0 g                                                    ______________________________________                                         *(3250.0 g on anhydrous basis)                                           

The blended material was milled through Fitz JT mill with a No. 2A plate(0.093") at slow speed with knives forward, the milled mixture blendedfor an additional 15 minutes, and weighed. The resulting milled andblended mixture (5977.2 g) was then blended for 5 minutes with a 9:1lubricant mixture of magnesium stearate:sodium lauryl sulfate (91.65 g),the further blend slugged on a Stokes DD-2 fitted with six stations of3/4" flat faced punches, and the slugs granulated by remilling andadditionally blending as specified above. Additional 9:1 lubricant (29.5g) was blended with the resulting granulated blend (5869 g) and thematerial encapsulated into #0 capsules on a Zanasi RM-63 capsule machineat a fill weight of 483±23 mg to yield capsules containing no more than275 mg and no less than the desired 250 mg of azithromycin activity.

By appropriately modifying the capsule size, the fill weight and theproportion of azithromycin in the blend, capsules containing 100 mg, 125mg, 375 mg or 500 mg of azithromycin activity are prepared.

4"-Epi-azithromycin, 4"-amino-4"-deoxy-azithromycin and4"-epi-4"-amino-4"deoxy-azithromycin capsules are prepared in likemanner, substituting in equal weight of the active ingredient (correctedfor potency as free base) for azithromycin.

EXAMPLE 4 Azithromycin Tablets (250 Mg) for Oral Administration

The following ingredients were accurately weighed, combined and blendedin a suitable blender for 30 minutes:

    ______________________________________                                        Azithromycin dihydrate                                                                           14245.0 g*                                                 Dibasic calcium phosphate                                                                        22205.0 g                                                  AC-DI-SOL          1620.0 g                                                   Magnesium stearate 1242.7 g                                                   ______________________________________                                         *(13,485.0 g on an anhydrous basis)                                      

The blend was milled in a Fitzpatrick D comminutor fitted with a No. 3plate (0.125") with knives forward at 3600 rpm, then blended for anadditional 30 minutes. To the resulting milled blend (39,192 g) wasadded an additional 783.8 g of magnesium stearate and blending continuedfor 5 minutes. The mixture was then slugged according to the precedingexample, and remilled as immediately above, and blended for 5 minutes.Additional magnesium stearate (394.5 g) was added to the resultinggranulated blend (39,445 g), blending was continued for 5 minutes, andthe mixture tableted on a Killian tableting machine with forced feederand 32"×5/8" upper and lower oval shaped punches, each tablet having aweight of 787 mg±37 mg, each containing no less than 250 mg and no morethan 275 mg of azithromycin activity.

EXAMPLE 5 Azithromycin for I.V. or I.M. Injection

In a sterile environment and using sterile, particle free equipment andcomponents, 10,949 g of water for injection was placed in a compoundingflask. Anhydrous citric acid, 494.4 g was added and dissolved withagitation. In a separate flask 310 g of sodium hydroxide was dissolvedin 690 g of water. A portion of the latter (755 g) was used to adjustthe pH of the citric acid from 1.63 to 5.09±0.02. Azithromycin dihydrate670.0 g (equivalent to 642.5 g of anhydrous base) was added, and themixture adjusted to pH 6.60±0.1 with 4.0 g additional of the sodiumhydroxide solution. Water (6076.5 g) was added to bring the resultingsolution to a final weight of 18,948.9 g. If desired, the solution issterile filtered at this stage, using a millipore filter. Using afilling machine, 50 ml flint type vials were each filled with 15.06±0.45g of this solution, loosely stoppered with gray teflon stoppers, andfreeze dried to yield stoppered vials each containing 51±1.5 mg ofazithromycin activity in the form of freeze dried solids. Prior to i.m.or i.v. injection, water for injection (10 ml) is added by injection bysyringe through the stopper, and the freeze dried solids redissolved byshaking. Virtually the entire contents of the vial is taken up into thesyringe and injected either i.v. or i.m.

PREPARATION 1 Hygroscopic Azithromycin Monohydrate

Substantially following the methylation procedure of Kobrehel et al.,U.S. Pat. No. 4,517,359; and the crystallization procedure of Bright,U.S. Pat. No. 4,474,768; 9-deoxo-9a-aza-9a-homoerythromycin A(previously called 11-aza-10-deoxo-10-dihydroerythromycin A; 100 g,0.218 mol) was dissolved with stirring in 400 ml CHCl₃. Formic acid(98%; 10.4 ml, 0.436 mol) and formaldehyde (37%; 16.4 ml, 0.349 mol)were added over 4-5 minutes, and the mixture heated at reflux for 20hours. The mixture was cooled to ambient temperature, diluted with 400ml H₂ O and adjusted to pH 10.5 with 50% NaOH. The aqueous layer wasseparated and extracted 2×100 ml with fresh CHCl₃. The organic layerswere combined, stripped in vacuo to 350 ml, twice diluted with 450 ml ofethanol and restripped to 350 ml, and finally diluted with 1000 ml H₂ Oover a 1 hour period, pausing for 15 minutes as a slurry began todevelop after the addition of about 250 ml of H₂ O. Title product wasrecovered by filtration and dried in air at 50° C. for 24 hours, 85 g;mp 136° C.; differential thermal analysis (heating rate 20° C./minute)shows an endotherm at 142° C.; thermal gravimetric analysis (heatingrate 30° C./minute) shows a 2.6% weight loss at 100° C. and a 4.5%weight loss at 150° C.; water content 3.92%; ethanol content 1.09%.

Anal. Calcd. for C₃₈ H₇₂ N₂ O₁₂ (corrected for ethanol and watercontent):

C, 58.46; H, 9.78; N, 3.74; Alkoxy, 4.67. Found: C, 58.40; H, 9.29; N,3.50; Alkoxy, 4.52.

A sample of the monohydrate (having a water content of 3.2%) wasmaintained at 18% relative humidity for 14 days. The sample lost waterover the first 24 hours to yield monohydrate having the theoreticalwater content (2.35%). The water content then remained substantiallyconstant over 14 days, a value of 2.26% being recorded at 14 days.

At 33% relative humidity the water content of a sample of the samemonohydrate rapidly rose to 5.6% where it remained substantially steadyfor at least three days. Similarly at 75% and 100% relative humidity,the water content rose rapidly, but was now maintained at even higherlevels, 6.6% and 7.2%, respectively, for at least 3 days.

I claim:
 1. A method of treating or preventing an infection in a mammal due to a strain of Toxoplasma gondii species which comprises administering to said mammal an anti-Toxoplasma gondii species effective amount of:azithromycin; 4"-epi-azithromycin; 4"-amino-4"-deoxy-azithromycin; or 4"-epi-4"-amino-4"-deoxy-azithromycin;or a pharmaceutically acceptable salt thereof.
 2. A method of claim 1 which comprises administering azithromycin, or a pharmaceutically acceptable salt thereof.
 3. A method of claim 1 which comprises administering 4"-epi-azithromycin or a pharmaceutically acceptable salt thereof.
 4. A method of claim 1 which comprises administering 4"-amino-4"-deoxy-azithromycin or a pharmaceutically acceptable salt thereof.
 5. A method of treating or preventing an infection in a mammal due to a strain of Toxoplasma gondii species which comprises administering to said mammal an anti-Toxoplasma gondii species effective amount of azithromycin dihydrate. 